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Distribution of somatostatin- and neuropeptide Y-immunoreactive nerve fibers in the porcine female reproductive system
Neuroscience Letters, 122 (1991) 273-276
273
Elsevier ScientificPublishers Ireland Ltd. NSL 07516
Distribution of somatostatin- and neuropeptide Y-immunoreactive nerve fibers in the porcine female reproductive system O. Hfipp61/i 1, M . L a k o m y 2, M . M a j e w s k i 2 a n d N . Y a n a i h a r a 3 1Department of Anatomy and Neurobiological Research Unit, University of Helsinki, Helsinki (Finland), 2Department of Anatomy, Veterinary Faculty, Academy of Agriculture and Technology, Olsztyn, Kortowo (Poland) and ~School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi (Japan)
(Received 14 May 1990; Revisedversion received 8 October 1990; Accepted 26 October 1990) Key words: Somatostatin; Neuropeptide Y; Female genital organ; Pig; Immunohistochemistry
The localization and distribution of somatostatin and neuropeptide Y were studied in the porcine female reproductive system with the indirect immunofluorescencetechnique. Somatostatin-immunoreactivenerve fibers were observed in different parts of the ovary and in the muscular membrane of the uterus as well as in the mesosalphinx. Somatostatin-immunoreactiveneurons were detected in the inferior mesentericganglion. Neuropeptide Y immunoreactivity was present in a large number of nerve fibers distributed in different regions of the uterus, oviduct and ovary. The present results suggest that the porcine female genital organs receive innervation by somatostatin- and neuropeptide Y-containing nerve fibers, but their exact functional role remains to be established.
Biochemical studies with radioimmunoassay technique have shown that somatostatin (SOM) exists in the ovary [8]. However, the localization and distribution of SOM in this organ has remained unknown, and previously there was no morphological evidence for the presence of SOM in neuronal elements of the female reproductive system o f any m a m m a l i a n species. The presence of neuropeptide Y (NPY) [1, 3, 5, 9, 10, 12] in female genital organs o f the rat, mouse, guinea pig and w o m a n has previously been well documented. In contrast, very little is known about the existence of N P Y in nerve fibers of female reproductive organs of breeding animals. In the pig, NPY-immunoreactive (IR) nerve fibers have been reported to exist in the ovary and oviduct [6], but not in the uterus. The aim of this study was to examine the localization and distribution of SOM and N P Y in neuronal elements of the porcine female genital organs with the indirect immunofluorescence technique. Fourteen pigs, average weight of 10 kg, were killed and the uterus, oviduct, ovaries as well as inferior mesenteric and paracervical ganglia were immediately removed and immersed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, p H 7.4, for 2 h, and then transferred to 0.1 M sodium phosphate buffer, p H 7.4, containing Correspondence: O. Hfipp61~i,Department of Anatomy and Neurobiological Research Unit, University of Helsinki, Siltavuorenpenger20 A, SF-00170 Helsinki, Finland.
0304-3940/91/$ 03.50 © 1991 ElsevierScientific Publishers Ireland Ltd.
20% sucrose for at least 24 h at 4°C. Ten p m cryostat sections were cut and mounted on chrome-alum-gelatin coated glass slides and processed for indirect immunohistochemistry as described earlier in detail [2]. The sections were incubated in the somatostatin (Amersham, code RPN. 1702 or IncStar, Lot 27092) or N P Y (Amersham, code RPN. 1612) antisera. Control sections were incubated in antisera which were preabsorbed with 1, 10 or 50 g M SOM or N P Y peptide, respectively. N P Y immunostaining was completely abolished with 1 p M N P Y peptide, while SOM immunoreactivity was abolished with 10 and 50 g M concentrations of somatostatin peptide. In this study no staining was observed if the specific antisera were omitted or if the sections were incubated in normal rabbit serum. In the ovary, a moderate number of S O M - I R varicose nerve fibers were found in cortical and medullary parts of the organ. The S O M - I R fibers were observed more frequenty in the cortical part of the ovary (Figs. 1 and 2). Delicate, moderately immunofluorescent nerve fibers were found in adventitial layers around blood vessels and close to follicles in different stages of development (Fig. 1). Some S O M - I R nerve fibers were localized in the thecal tunics (Fig. 1) and in medullary part of the ovary (Fig. 3). Only a few S O M - I R delicate, moderately immunofluorescent nerve fibers were detected in the uterine horns (Fig. 4). These fibers were seen in the myometrial layers between the smooth muscle cells (Fig. 4). Bundles
274
Fig. 1. SOM-IR nerve terminals close to ovarian follicles. Bar in Figs. 1 6 = 30/~m. Fig. 2. SOM-IR nerve fiber (arrows) in cortical part of the pig ovary, with no relation to follicles. Fig. 3. A SOM-IR nerve fiber around a blood vessel in the medullary part of the ovary.
Fig. 4. SOM-IR nerve fibers (arrows) in the circular layer of myometrium. Fig. 5. Two SOM-IR principal neurons in the inferior mesenteric ganglion. Fig. 6. A SOM-IR nerve bundle in the mesosalphinx.
o f intensely SOM'-IR nerve fibers were also observed in the mesosalphinx (Fig. 6). In order to find out the possible origin o f these nerve fibers, the presence and distribution o f S O M I R was also studied in the preaortic inferior mesenteric ganglion ( I M G ) and in paracervical ganglia (PG). A minor subpo-
pulation o f neurons in the I M G , but not in PG, exhibited S O M I R (Fig. 5). The uterine horns contained a great n u m b e r o f N P Y I R nerve fibers, located in each m e m b r a n e of the organ. In the subserous layer intense N P Y - I R nerve fibers or bundles (Fig. 7) were seen, which were often localized
275
Fig. 7. A bundle of NPY-IR nerve fibers in the subserous layer of uterine horn. Bar in the Figs. 7-I 0 = 30 gm.
Fig. 8. NPY-IR nerve terminals in close relation to a blood vessel in the uterine horn. Fig. 9. Numerous NPY-IR nerve fibers running parallel to muscular bundles in the uterine horn. Fig. 10. NPY-IR nerve fibers in the endometrium.
around arterial blood vessels, mainly in the region of attachment of the broad uterine ligament (Fig. 8). Nerve fibers running parallel to the long axis of smooth muscle cells were seen in circular layer o f the myometrium (Fig. 9). A large number of N P Y - I R varicose nerve fibers and bundles were also localized under the mucosal membrane of the uterus (Fig. 10) and in different regions of the oviduct and ovary. Previously, there was no evidence for the existence of SOM-containing nerve fibers in the female genital organs of any m a m m a l i a n species. Earlier studies, how-
ever, have supported the concept that the rat ovary, and the uterus of w o m a n and guinea pig do not contain S O M - I R nerve fibers [3, 7]. On the other hand, previous biochemical evidence has indicated that SOM is present in the ovary of the pig [8]. The results o f this study are in accordance with these biochemical observations and indicate that the porcine ovaries and uterus receive innervation by SOM-containing nerve fibers. According to our observations, nerve bundles of intense S O M - I R fibers were also found to be present in the wide uterine ligament, namely mesosalphinx, suggesting that these
276 nerve bundles m a y supply S O M - I R fibers to the female genital organs. The functional role o f S O M - I R nerve fibers in these organs remains u n k n o w n . Previous biochemical studies, however, suggest that S O M m a y have a role in m a t u r a t i o n o f oocytes in porcine ovary [8]. A population o f sympathetic neurons in the preaortic coeliac-superior mesenteric ganglion and in the I M G have previously been shown to contain S O M I R [4]. Moreover, McNeill and Burden [7] have provided evidence that in the rat, S O M - I R neurons located in preaortic ganglia, including the I M G , project to the ovary. In this study a m i n o r population o f principal neurons in the I M G , but not those in the PG, contained S O M IR, supporting the possibility that some o f the S O M - I R nerve fibers in the porcine female genital organs, particularly those in the ovary, m a y originate f r o m a population o f S O M - I R principal neurons o f the I M G . The results o f this study indicate that n u m e r o u s N P Y I R nerve fibers are present in the porcine uterus and they are distributed in subserous, myometrial and submucous layers. The present observations concerning the localization and distribution o f N P Y in the porcine uterus are consistent with previous data on other species [1, 3, 5, 9, 10, 12]. In the genitourinary tract, N P Y has previously been shown e.g. to inhibit noradrenaline [11] release in the h u m a n fallopian tube, and acetylcholine release in the rat cervix uteri [12]. The skillful technical assistance by Ms. Marja-Leena Piironen, Ms. Maija-Leena J o h a n s s o n and Mr. Reijo Karppinen is gratefully acknowledged. This study was supported by grants f r o m the Paulo F o u n d a t i o n (O.H.) and partly by the Polish Ministry o f N a t i o n a l Education, M R II-10 (M.L.). 1 Fried, G., H6kfelt, T., Terenius, S. and Goldstein, M.., Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced
during pregnancy in parallel with noradrenergic nerves, Histochemistry, 83 (1985)437-442. 2 Hhpp61fi, O., Soinila, S., P~iiv~irinta, H. and Panula, P., [Met5] enkephalin-Arg6-Phe7- and [Mets] enkephalin-Arg6-GlyT-Leu8immunoreactive nerve fibers and neurons in the superior cervical ganglion of the rat, Neuroseience, 21 (1987) 283-295. 3 Heinrich, D., Reinecke, M. and Forssman, W.G., Peptidergic innervation of the human and guinea pig uterus, Arch. Gynecol., 237 (1986) 213-219. 4 H6kfelt, T., Elfvin, L.G., Elde, R., Schultzberg, M., Goldstein, M. and Luft, R., Occurrence of somatostatin-like immunoreactivity in some peripheral sympathetic noradrenergic neurons, Proc. Natl. Acad. Sci., U.S.A., 79 (1977) 358%3591. 5 Huang, W.M., Gu, J., Blank, M.A., Allen, J.M., Bloom, S.R. and Polak, J.M., Peptide-immunoreactive nerves in the mammalian female genital tract, Histochem. J., 16 (1984) 1297-1310. 6 Kannisto, P., Ekblad, E., Helm, G., Owman, Ch., Sj6berg, N.O., Stjernquist, F., Sundler, F. and Walles, B., Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry, Histochemistry, 86 (1986) 25-34. 7 McNeill, D.L. and Burden, H.W., Neuropeptide Y and somatostatin immunoreactive perikarya in preaortic ganglia projecting to the rat ovary, J. Reprod. Fertil., 78 (1986) 727-732. 8 Moil, T., Suito, H., Ohno, Y., Ianahara, M., Mosoi, E. and Saito, S., Evidence for the existence of somatostatin-like immunoreactivity with molecular heterogeneity in porcine ovaries, Acta Endocrinol., 106 (1984) 254-259, 9 Papka, R.E. and Traurig, H.H., Distribution of subgroups of neuropeptide Y-immunoreactive and noradrenergic nerves in the female rat uterine cervix, Cell Tissue Res., 252 (1988) 533-541. 10 Papka, R.E., Cotton, J,P. and Traurig, H.H., Comparative distribution of neuropeptide tyrosine, VIP, substance P-immunoreactive, acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat, Cell Tissue Res., 242 (1985) 475-490. 11 Samuelsson, U.E. and Dalsgaard, C.J., Action and localization of neuropeptide Y in the human Fallopian tube, Neurosci. Lett., 58 (1985) 49-54. 12 Stjernquist, M., Emson, P., Owman, C., Sj6berg, N.-O., Sundler, F. and Tatemoto, K., Neuropeptide Y in the female reproductive tract of the rat, Distribution of nerve fibers and motor effects, Neurosci. Lett., 39 (1983) 279-284.
273
Elsevier ScientificPublishers Ireland Ltd. NSL 07516
Distribution of somatostatin- and neuropeptide Y-immunoreactive nerve fibers in the porcine female reproductive system O. Hfipp61/i 1, M . L a k o m y 2, M . M a j e w s k i 2 a n d N . Y a n a i h a r a 3 1Department of Anatomy and Neurobiological Research Unit, University of Helsinki, Helsinki (Finland), 2Department of Anatomy, Veterinary Faculty, Academy of Agriculture and Technology, Olsztyn, Kortowo (Poland) and ~School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi (Japan)
(Received 14 May 1990; Revisedversion received 8 October 1990; Accepted 26 October 1990) Key words: Somatostatin; Neuropeptide Y; Female genital organ; Pig; Immunohistochemistry
The localization and distribution of somatostatin and neuropeptide Y were studied in the porcine female reproductive system with the indirect immunofluorescencetechnique. Somatostatin-immunoreactivenerve fibers were observed in different parts of the ovary and in the muscular membrane of the uterus as well as in the mesosalphinx. Somatostatin-immunoreactiveneurons were detected in the inferior mesentericganglion. Neuropeptide Y immunoreactivity was present in a large number of nerve fibers distributed in different regions of the uterus, oviduct and ovary. The present results suggest that the porcine female genital organs receive innervation by somatostatin- and neuropeptide Y-containing nerve fibers, but their exact functional role remains to be established.
Biochemical studies with radioimmunoassay technique have shown that somatostatin (SOM) exists in the ovary [8]. However, the localization and distribution of SOM in this organ has remained unknown, and previously there was no morphological evidence for the presence of SOM in neuronal elements of the female reproductive system o f any m a m m a l i a n species. The presence of neuropeptide Y (NPY) [1, 3, 5, 9, 10, 12] in female genital organs o f the rat, mouse, guinea pig and w o m a n has previously been well documented. In contrast, very little is known about the existence of N P Y in nerve fibers of female reproductive organs of breeding animals. In the pig, NPY-immunoreactive (IR) nerve fibers have been reported to exist in the ovary and oviduct [6], but not in the uterus. The aim of this study was to examine the localization and distribution of SOM and N P Y in neuronal elements of the porcine female genital organs with the indirect immunofluorescence technique. Fourteen pigs, average weight of 10 kg, were killed and the uterus, oviduct, ovaries as well as inferior mesenteric and paracervical ganglia were immediately removed and immersed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, p H 7.4, for 2 h, and then transferred to 0.1 M sodium phosphate buffer, p H 7.4, containing Correspondence: O. Hfipp61~i,Department of Anatomy and Neurobiological Research Unit, University of Helsinki, Siltavuorenpenger20 A, SF-00170 Helsinki, Finland.
0304-3940/91/$ 03.50 © 1991 ElsevierScientific Publishers Ireland Ltd.
20% sucrose for at least 24 h at 4°C. Ten p m cryostat sections were cut and mounted on chrome-alum-gelatin coated glass slides and processed for indirect immunohistochemistry as described earlier in detail [2]. The sections were incubated in the somatostatin (Amersham, code RPN. 1702 or IncStar, Lot 27092) or N P Y (Amersham, code RPN. 1612) antisera. Control sections were incubated in antisera which were preabsorbed with 1, 10 or 50 g M SOM or N P Y peptide, respectively. N P Y immunostaining was completely abolished with 1 p M N P Y peptide, while SOM immunoreactivity was abolished with 10 and 50 g M concentrations of somatostatin peptide. In this study no staining was observed if the specific antisera were omitted or if the sections were incubated in normal rabbit serum. In the ovary, a moderate number of S O M - I R varicose nerve fibers were found in cortical and medullary parts of the organ. The S O M - I R fibers were observed more frequenty in the cortical part of the ovary (Figs. 1 and 2). Delicate, moderately immunofluorescent nerve fibers were found in adventitial layers around blood vessels and close to follicles in different stages of development (Fig. 1). Some S O M - I R nerve fibers were localized in the thecal tunics (Fig. 1) and in medullary part of the ovary (Fig. 3). Only a few S O M - I R delicate, moderately immunofluorescent nerve fibers were detected in the uterine horns (Fig. 4). These fibers were seen in the myometrial layers between the smooth muscle cells (Fig. 4). Bundles
274
Fig. 1. SOM-IR nerve terminals close to ovarian follicles. Bar in Figs. 1 6 = 30/~m. Fig. 2. SOM-IR nerve fiber (arrows) in cortical part of the pig ovary, with no relation to follicles. Fig. 3. A SOM-IR nerve fiber around a blood vessel in the medullary part of the ovary.
Fig. 4. SOM-IR nerve fibers (arrows) in the circular layer of myometrium. Fig. 5. Two SOM-IR principal neurons in the inferior mesenteric ganglion. Fig. 6. A SOM-IR nerve bundle in the mesosalphinx.
o f intensely SOM'-IR nerve fibers were also observed in the mesosalphinx (Fig. 6). In order to find out the possible origin o f these nerve fibers, the presence and distribution o f S O M I R was also studied in the preaortic inferior mesenteric ganglion ( I M G ) and in paracervical ganglia (PG). A minor subpo-
pulation o f neurons in the I M G , but not in PG, exhibited S O M I R (Fig. 5). The uterine horns contained a great n u m b e r o f N P Y I R nerve fibers, located in each m e m b r a n e of the organ. In the subserous layer intense N P Y - I R nerve fibers or bundles (Fig. 7) were seen, which were often localized
275
Fig. 7. A bundle of NPY-IR nerve fibers in the subserous layer of uterine horn. Bar in the Figs. 7-I 0 = 30 gm.
Fig. 8. NPY-IR nerve terminals in close relation to a blood vessel in the uterine horn. Fig. 9. Numerous NPY-IR nerve fibers running parallel to muscular bundles in the uterine horn. Fig. 10. NPY-IR nerve fibers in the endometrium.
around arterial blood vessels, mainly in the region of attachment of the broad uterine ligament (Fig. 8). Nerve fibers running parallel to the long axis of smooth muscle cells were seen in circular layer o f the myometrium (Fig. 9). A large number of N P Y - I R varicose nerve fibers and bundles were also localized under the mucosal membrane of the uterus (Fig. 10) and in different regions of the oviduct and ovary. Previously, there was no evidence for the existence of SOM-containing nerve fibers in the female genital organs of any m a m m a l i a n species. Earlier studies, how-
ever, have supported the concept that the rat ovary, and the uterus of w o m a n and guinea pig do not contain S O M - I R nerve fibers [3, 7]. On the other hand, previous biochemical evidence has indicated that SOM is present in the ovary of the pig [8]. The results o f this study are in accordance with these biochemical observations and indicate that the porcine ovaries and uterus receive innervation by SOM-containing nerve fibers. According to our observations, nerve bundles of intense S O M - I R fibers were also found to be present in the wide uterine ligament, namely mesosalphinx, suggesting that these
276 nerve bundles m a y supply S O M - I R fibers to the female genital organs. The functional role o f S O M - I R nerve fibers in these organs remains u n k n o w n . Previous biochemical studies, however, suggest that S O M m a y have a role in m a t u r a t i o n o f oocytes in porcine ovary [8]. A population o f sympathetic neurons in the preaortic coeliac-superior mesenteric ganglion and in the I M G have previously been shown to contain S O M I R [4]. Moreover, McNeill and Burden [7] have provided evidence that in the rat, S O M - I R neurons located in preaortic ganglia, including the I M G , project to the ovary. In this study a m i n o r population o f principal neurons in the I M G , but not those in the PG, contained S O M IR, supporting the possibility that some o f the S O M - I R nerve fibers in the porcine female genital organs, particularly those in the ovary, m a y originate f r o m a population o f S O M - I R principal neurons o f the I M G . The results o f this study indicate that n u m e r o u s N P Y I R nerve fibers are present in the porcine uterus and they are distributed in subserous, myometrial and submucous layers. The present observations concerning the localization and distribution o f N P Y in the porcine uterus are consistent with previous data on other species [1, 3, 5, 9, 10, 12]. In the genitourinary tract, N P Y has previously been shown e.g. to inhibit noradrenaline [11] release in the h u m a n fallopian tube, and acetylcholine release in the rat cervix uteri [12]. The skillful technical assistance by Ms. Marja-Leena Piironen, Ms. Maija-Leena J o h a n s s o n and Mr. Reijo Karppinen is gratefully acknowledged. This study was supported by grants f r o m the Paulo F o u n d a t i o n (O.H.) and partly by the Polish Ministry o f N a t i o n a l Education, M R II-10 (M.L.). 1 Fried, G., H6kfelt, T., Terenius, S. and Goldstein, M.., Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced
during pregnancy in parallel with noradrenergic nerves, Histochemistry, 83 (1985)437-442. 2 Hhpp61fi, O., Soinila, S., P~iiv~irinta, H. and Panula, P., [Met5] enkephalin-Arg6-Phe7- and [Mets] enkephalin-Arg6-GlyT-Leu8immunoreactive nerve fibers and neurons in the superior cervical ganglion of the rat, Neuroseience, 21 (1987) 283-295. 3 Heinrich, D., Reinecke, M. and Forssman, W.G., Peptidergic innervation of the human and guinea pig uterus, Arch. Gynecol., 237 (1986) 213-219. 4 H6kfelt, T., Elfvin, L.G., Elde, R., Schultzberg, M., Goldstein, M. and Luft, R., Occurrence of somatostatin-like immunoreactivity in some peripheral sympathetic noradrenergic neurons, Proc. Natl. Acad. Sci., U.S.A., 79 (1977) 358%3591. 5 Huang, W.M., Gu, J., Blank, M.A., Allen, J.M., Bloom, S.R. and Polak, J.M., Peptide-immunoreactive nerves in the mammalian female genital tract, Histochem. J., 16 (1984) 1297-1310. 6 Kannisto, P., Ekblad, E., Helm, G., Owman, Ch., Sj6berg, N.O., Stjernquist, F., Sundler, F. and Walles, B., Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry, Histochemistry, 86 (1986) 25-34. 7 McNeill, D.L. and Burden, H.W., Neuropeptide Y and somatostatin immunoreactive perikarya in preaortic ganglia projecting to the rat ovary, J. Reprod. Fertil., 78 (1986) 727-732. 8 Moil, T., Suito, H., Ohno, Y., Ianahara, M., Mosoi, E. and Saito, S., Evidence for the existence of somatostatin-like immunoreactivity with molecular heterogeneity in porcine ovaries, Acta Endocrinol., 106 (1984) 254-259, 9 Papka, R.E. and Traurig, H.H., Distribution of subgroups of neuropeptide Y-immunoreactive and noradrenergic nerves in the female rat uterine cervix, Cell Tissue Res., 252 (1988) 533-541. 10 Papka, R.E., Cotton, J,P. and Traurig, H.H., Comparative distribution of neuropeptide tyrosine, VIP, substance P-immunoreactive, acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat, Cell Tissue Res., 242 (1985) 475-490. 11 Samuelsson, U.E. and Dalsgaard, C.J., Action and localization of neuropeptide Y in the human Fallopian tube, Neurosci. Lett., 58 (1985) 49-54. 12 Stjernquist, M., Emson, P., Owman, C., Sj6berg, N.-O., Sundler, F. and Tatemoto, K., Neuropeptide Y in the female reproductive tract of the rat, Distribution of nerve fibers and motor effects, Neurosci. Lett., 39 (1983) 279-284.